Composition and method for treatment of MRSA

ABSTRACT

The present invention provides a photosensitizing composition for treatment of MRSA comprising a photosensitizer and chlorhexidine and a pharmaceutically acceptable carrier. The present invention also provides a method for reducing disease causing microbes comprising: applying the composition comprising a photosensitizer, chlorhexidine at a concentration of more than about 0.01% and less than about 2% v/v, and a pharmaceutically acceptable carrier to a treatment site; and applying light to the treatment site at a wavelength absorbed by the photosensitizer so as to reduce the microbes at the treatment site.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.12/512,295, filed on Jul. 30, 2009, which claims benefit of provisionalapplication Ser. No. 61/085,577 filed on Aug. 1, 2008 and provisionalapplication Ser. No. 61/186,068, filed Jun. 11, 2009, which are alltitled: “COMPOSITION AND METHOD FOR TREATMENT OF MRSA” and are herebyentirely incorporated by reference for all purposes.

FIELD OF INVENTION

The present invention provides a photosensitizing composition and aphotodynamic disinfection method using such composition for treatment ofMethicillin-resistant Staphylococcus aureus (“MRSA”) that enhance MRSAtreatment efficacy while reducing irritation and sensitivity to the hosttissues at the treatment site.

BACKGROUND OF THE INVENTION

MRSA, a spherical Gram-positive aerobe, accounts for up to 50% ofnosocomial S. aureus infections, and represents a multi-billion dollarproblem in critical care units, intensive care units and generalhospitals worldwide. Because bacteria naturally adapt to antibiotics,more than 95% of patients with MRSA do not respond to first-lineantibiotics. Certain MRSA strains are now even resistant to glycopeptideantibiotics like Vancomycin®, removing the last remaining effectiveantibiotic treatment for the disease. Due to the fact that MRSA isresistant to most antibiotics such as methicillin, oxacillin, penicillinand amoxicillin, there is a need to treat MRSA without the use ofantibiotics.

Photodynamic disinfection is a desirable alterative treatment method asit has been demonstrated to be an effective non-antibiotic antimicrobialapproach in vitro. One exemplary advantage of photodynamic disinfectionas a MRSA treatment modality is that, due to this non-specificbactericidal mechanism, it is typically not subject to issues ofresistance that can plague the use of antibiotics. As another exemplaryadvantage, it can be employed as a localized topical treatment that canbe administered in areas such as the nasal cavities (e.g., nasal mucosa)where MRSA is mostly likely found in the human body.

Photodynamic disinfection fundamentally involves the use of light energyto activate one or more photosensitizers of a photosensitizingcomposition so that those photosensitizers can then either pass energyon directly to a substrate/target (type I reaction), or can interactwith molecular oxygen to produce reactive oxygen species (type IIreaction). These reactions mediate the non-specific reduction of MRSAand other microbial cells primarily via lipid peroxidation, membranedamage, and damage to intracellular components.

SUMMARY OF THE INVENTION

The present invention provides a photosensitizing composition fortreatment of MRSA comprising a photosensitizer (e.g., phenothiazine) andchlorhexidine and a pharmaceutically acceptable carrier. As shown below,this composition when used for photodynamic disinfection of MRSAenhances MRSA treatment efficacy. Furthermore, in one embodiment of thepresent invention, the photosensitizing composition also reduces and/oreliminates irritation and sensitivity to host tissues at the treatmentsite.

The present invention also provides a method for treatment of MRSAcomprising: applying the composition comprising a photosensitizer,chlorhexidine and a pharmaceutically acceptable carrier to a treatmentsite; and applying light to the treatment site at a wavelength absorbedby the photosensitizer so as to reduce MRSA at the treatment site.

The present invention further provides a method for reducing diseasecausing microbes comprising: applying a composition comprising aphotosensitizer, chlorhexidine at a concentration of more than about0.01% and less than about 2% v/v, and a pharmaceutically acceptablecarrier to a treatment site containing disease causing microbes; andapplying light to the treatment site at a wavelength absorbed by thephotosensitizer so as to reduce the microbes at the treatment site.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and inventive aspects of the present invention will becomemore apparent upon reading the following detailed description, claims,and drawings, of which the following is a brief description:

FIG. 1 is a graph showing the absorbance profile of three compositionsdescribed below in Example I; and

FIG. 2 is a table showing the data collected for the experimentsdescribed below in Example II.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In the present invention, chlorhexidine is combined with aphotosensitizer to increase the effects of photodynamic disinfection toreduce, eliminate and/or kill (hereinafter collectively refer to as“reduce”, “reducing”, and/or “reduction”) disease causing microbes suchas MRSA or the like. The photosensitizing composition of the presentinvention includes a photosensitizer, chlorhexidine and apharmaceutically acceptable carrier. As discussed below, the compositioncombines the powerful short-term antimicrobial effects of photodynamicdisinfection with a more sustained chemical disinfection provided bychlorhexidine.

Chlorhexidine (e.g., chlorhexidine gluconate, chlorhexidine digluconate,chlorhexidine dihydrochloride, chlorhexidine diacetate or the like) is abroad spectrum antiseptic used for topical skin surface disinfection(e.g., surgical scrub or the like). For such application, chlorhexidineis commonly used in concentrations at ≧2 percentage of total volume (“%v/v”). See e.g., BactoShield® (2%, 4%), Betasept® (4%), ChloraPrep®(2%), Chlorostat®: (2%), Dial® Surgical Scrub (4%), Dyna-Hex (2%, 4%)Hibiclens®: (4%) and Operand® (2%). Irritation and sensitivity have beenreported with such use of chlorhexidine containing products, especiallyin sensitive skin areas.

In one embodiment of the present invention, chlorhexidine is provided ata concentration that reduces and/or eliminates potential irritation andsensitivity to host tissues at the treatment area. This reduction and/orelimination of potential irritation and sensitivity is especiallyhelpful when the host tissues at the treatment area are sensitivetissues such as the nasal mucosa. Exemplary suitable concentrations areabout 1% v/v; about 0.5% v/v; about 0.25% v/v; about 0.125% v/v; betweenabout 0.125% v/v and about 1% v/v; between about 0.125% v/v and about0.8% v/v; between about 0.125% v/v and about 1.5% v/v; between about0.25% v/v and about 0.5% v/v; between about 0.25% v/v and about 1% v/v;between about 0.25% v/v and about 1.5% v/v; a range that is less thanabout 1% v/v but more than about 0.1% v/v; a range that is less thanabout 0.8% v/v but more than about 0.1% v/v; a range that is less thanabout 2% v/v but more than about 0.1% v/v; and a range that is less thanabout 2% v/v but more than about 0.125%. The term “about” as used hereinin this specification shall mean+/−20% of the stated value.

Examples of the photosensitizer include photosensitizers that effectboth Type I and Type II photoreactions, where Type I reactions produceelectron abstraction redox-type reactions upon the application of lightand Type II reactions produce singlet oxygen (via molecular oxygen) uponthe application of light. Suitable classes of compounds that may be usedas the photosensitizer include tetrapyrroles or derivatives thereof suchas porphyrins, chlorins, bacteriochlorins, phthalocyanines,naphthalocyanines, texaphyrins, verdins, purpurins or pheophorbides,phenothiazines, etc., such as those described in U.S. Pat. Nos.6,211,335; 6,583,117; and 6,607,522 and U.S. Patent Publication No.2003-0180224. Preferred phenothiazines include methylene blue, toluidineblue, and those discussed in U.S. Patent Publication No. 2004-0147508.Another preferred photosensitizer is indocyanine green. The presentinvention also contemplates the use of two or more photosensitizers,such as methylene blue and toluidine blue or the like. Thephotosensitizers mentioned above are examples and are not intended tolimit the scope of the present invention in any way.

The photosensitizer may be present in the photosensitizing compositionin any suitable amounts. Examples are between about 0.001 percentage oftotal weight (% wt) and about 10% wt, between about 0.005% wt and about5% wt, between about 0.01% wt to about 1% wt, between about 0.01% wt toabout 0.1% wt, and no more than about 1% wt. The percentage of totalweight (% wt) can also be converted to percentage of total weight tovolume (% w/v) or percentage of total volume to volume (% v/v). For thepurpose of this specification, the concentration of photosensitizer canbe expressed either in % wt, % w/v, or % v/v and such expression ofconcentration is intended to include its equivalences (e.g., ifexpressed in % wt, it is intended include the equivalent concentrationmeasured in % w/v and % v/v).

As shown in Example II below, chlorhexidine significantly enhancedantimicrobial efficacy of photodynamic disinfection in reducing and/oreliminating microbial pathogens such as MRSA, even at low concentrationlevels such as between about 0.1% v/v and about 1% v/v. At chlorhexidineconcentrations between 0.125% v/v and 0.5% v/v, the antibacterialactivity of chlorhexidine and photodynamic disinfection combined isgreater than would be expected considering just the additive effects ofthe two antibacterial methods on their own. This indicates an unexpectedpotentiation of antibacterial effect when low concentration ofchlorhexidine and photodynamic disinfection are deliveredsimultaneously. This potentiation even occurs when chlorhexidine is usedat a lower concentration than what is normally used for conventionaltopical skin disinfection. Thus, the lower concentration ofchlorhexidine both reduces and/or eliminates irritation and sensitivitynormally associated with chlorhexidine, and still acts to increase theantibacterial ability of the photodynamic reaction. This is especiallyimportant in the treatment of MRSA located in the nasal cavity due tothe sensitivity of the nasal mucosa as a treatment site and the need toeradicate all MRSA pathogenic organisms to prevent recolonization.

The photosensitizing composition of the present invention furtherincludes a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier is a diluent, adjuvant, excipient, or vehicle withwhich the other components (e.g., the photosensitizer and thechlorhexidine, etc.) of the composition are administered. Thepharmaceutically acceptable carrier is preferably approved by aregulatory agency of the Federal or a state government, or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The pharmaceuticallyacceptable carriers are preferably sterile liquids. Examples of thepharmaceutically acceptable carriers include but are not limited towater, saline solution, dextrose solution, glycerol solution, phosphatebuffered saline solution, etc.

It is further preferred that the pharmaceutically acceptable carrier,when combined with the photosensitizer and the chlorhexidine, allows thephotosensitizing composition to have a viscosity low enough to flow intothe treatment site while also having a viscosity high enough to maintainthe composition within the treatment site. Further compositions thatbecome liquid after application to the treatment site are contemplatedsuch as those that melt or go into solution in the treatment site.Alternately, the composition may gel after application to the treatmentsite as a liquid; this would permit the composition to cover thetreatment site effectively, while also maintaining the composition inthe treatment site.

The present invention also provides a photodynamic disinfection methodfor treatment of MRSA comprising: applying the photosensitizingcomposition of the present invention described above to a treatmentsite; and applying light to the treatment site at a wavelength absorbedby the photosensitizing composition so as to reduce MRSA at thetreatment site. The treatment site for the method of the presentinvention to treat MRSA would preferably be the nasal cavity (e.g.,nasal mucosa) as it is generally known as an active site for MRSA.Photodynamic disinfection of the anterior nares of the nasal cavityreduces and/or eliminates MRSA.

It is preferred that prior to the application of light to the treatmentsite, the photosensitizing composition is placed into contact with thetreatment site for at least about 1 second, more preferably for at leastabout 5 seconds, even more preferably for at least about 10 seconds, andmost preferably from about 10 seconds to 30 seconds.

The light to be applied during the method of the present invention canbe at any wavelength(s) that can be absorbed by the photosensitizer(s)contained in the photosensitizing composition. The wavelengths aregenerally between about 160 nm to 1600 nm, between about 400 nm to about900 nm, and between about 500 nm to about 850 nm, although thewavelengths may vary depending upon the particular photosensitizingcompound used and the light intensity. For example, if thephotosensitizer is methylene blue, then the wavelength is preferablyranged from about 650 nm to 685 nm, more preferably from about 660 nm toabout 680 nm, and most preferably at about 665 nm to about 675 nm.

The light produced may be a single wavelength or multiple wavelengths.The light may be produced by any suitable art-disclosed light emittingdevices such as lasers, light emitting diodes (“LEDs”), incandescentsources, fluorescent sources, or the like. It is preferred that thelight is produced either by a laser or LEDS.

Depending on the photosensitizer concentration and the power of thelight emitting device(s), the application of light to the treatment sitemay only require a short period of time such as from about 15 seconds toless than about 5 minutes, preferably from about 15 seconds to about twominutes, more preferably for about 15 seconds to about 90 seconds, andmost preferably for about 30 seconds to 60 seconds. The light energyprovided during each cycle of application of light is preferred to rangefrom about 1 J/cm² to about 25 J/cm², more preferably at about 5 J/cm²to about 20 J/cm², and most preferably at about 6 J/cm² to about 12J/cm². Depending on the nature and extent of the MRSA located at thetreatment site, the practitioner may apply multiple cycles of lightapplications (e.g., about 2 to about 10, about 3 to about 5, etc.) tothe treatment site thereby resulting in a total accumulated light energyapplied to treatment site that can be substantially higher than thelight energy provided during each cycle. Again depending on the natureand extent of the microbes located at the treatment site, the entiremethod can be repeated multiple times (e.g., about 2 to about 10, about3 to about 5, etc.) until the desired effects have been reached. It ispreferred that the selections of photosensitizer concentration,wavelength, and/or total accumulated light energy applied to treatmentsite will allow the method of the present invention to reduce over about90%, more preferably over 95%, and most preferably over 99% of thetarget MRSA at the treatment site. It is also preferred that theapplication of light to the treatment site does not cause physiologicaldamage to the host tissues at and/or surround the treatment site.

The photosensitizing composition and the photodynamic disinfectionmethod of the present invention discussed above can also be used toreduce other disease-related microbes such as virus, fungus, andbacteria. Some examples of such microbes include but are not limited to,Staphylococcus aureus, Escherichia coli (“E. coli”), Enterococcusfaecalis (“E. faecalis”), Pseudomonas aeruginosa, Aspergillus, Candida,Clostridium difficile, Staphylococcus epidermidis, Acinetobacter sp.,and pathogenic Gram negative organisms generally residing within theoral cavity (e.g., Porphyromonas, Prevotella, Fusobacterium, Tannerella,Actinobacillus, Selenomonas, Eikenella, Campylobacter, Wolinella, etc.).

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes.

The following examples provided in accordance to the present inventionare for illustrative purpose only and are not intended as beingexhaustive or limiting of the invention.

Example I

Referring to FIG. 1, the characteristic absorbance profiles of thefollowing three compositions are provided: (a) methylene blue at aconcentration of 0.01% wt in pure water; (b) chlorhexidine gluconate ata concentration of 0.5% v/v in pure water; and (c) methylene blue at aconcentration of 0.01% wt and chlorhexidine gluconate at a concentrationof 0.5% v/v in pure water. The horizontal scale of FIG. 1 shows theabsorbance per unit length (i.e., optical density). The vertical scaleof FIG. 1 shows the wavelength in nm. The three lines (a, b, and c) inFIG. 1 represent the absorbance profiles of these three compositions.The characteristic absorbance profiles shown in FIG. 1 indicate that theaddition of the 0.5% v/v chlorhexidine gluconate to the 0.01% wtmethylene blue composition does not significantly alter the absorbancecharacteristics of the methylene blue in the visible wavelength range.

Example II

In vitro experiments were conducted by applying controls as describedbelow and several different combinations of chlorhexidine digluconateand methylene blue compositions to planktonic cultures of MRSA(Methicillin-resistant Staphylococcus aureus ATCC® 33592™) atapproximately 10⁷ CFU/ml. As shown in FIG. 2, these combinationsconsisted of the following active ingredients (a) methylene blue at aconcentration of 0.01% wt and chlorhexidine gluconate at a concentrationof 0.001% v/v; (b) methylene blue at a concentration of 0.01% wt andchlorhexidine gluconate at a concentration of 0.01% v/v; (c) methyleneblue at a concentration of 0.01% wt and chlorhexidine gluconate at aconcentration of 0.125% v/v; (d) methylene blue at a concentration of0.01% wt and chlorhexidine gluconate at a concentration of 0.25% v/v;and (e) methylene blue at a concentration of 0.01% wt and chlorhexidinegluconate at a concentration of 0.5% v/v. Also shown in FIG. 2, thecontrol formulations were consisted of (f) methylene blue at aconcentration of 0.01% wt alone; (g) chlorhexidine gluconate at aconcentration of 0.001% v/v alone; (h) chlorhexidine gluconate at aconcentration of 0.125% v/v alone; (i) chlorhexidine gluconate at aconcentration of 0.25% v/v alone; and (j) chlorhexidine gluconate at aconcentration of 0.5% v/v alone. All of the above-mentioned planktonicMRSA cultures were irradiated by a non-thermal diode laser with 220 mWpower output at a wavelength of 670 nm for 30 seconds (energy dose=10.3Joules/cm²).

Thereafter, all of the planktonic MRSA cultures were examined and dataregarding the amounts of planktonic MRSA reductions were collected. InFIG. 2, the composition number as discussed above is shown in column“I”; the concentration of methylene blue in each of the cultures isshown in column “II”; the concentration of chlorhexidine gluconate ineach of the cultures is shown in column “III”; and the reduction inviability of planktonic MRSA (expressed as log₁₀ reduction in viablecolony count vs. non-treated control) for each of the cultures comparedto a planktonic MRSA culture in purified water without any irradiation(“Control”) is shown in column “IV”. The rows in FIG. 2 show the resultof each of the cultures discussed above. As shown in FIG. 2, thereduction in MRSA viability obtained using methylene blue alone at aconcentration of 0.01% wt (see row “f”) was 3.1 log₁₀ compared to theControl, while the reductions of MRSA viability obtained after exposureto the chlorhexidine gluconate alone compositions (see rows “g”, “h”,“l”, and “j”) were between 0 to 2.7 log₁₀ (depending on thechlorhexidine gluconate's concentration) compared to the Control. Thedata showed that the reduction in MRSA viability obtained after exposureto the methylene blue and chlorhexidine gluconate combined compositionsin the presence of light corresponded to 100% eradication (>7.2 log₁₀reduction) when the chlorhexidine gluconate concentration was at either0.25% v/v or 0.5% v.v. When the chlorhexidine gluconate concentrationwas at 0.125% v/v, the reduction in MRSA viability was >99.999% (5.7log₁₀ reduction). At chlorhexidine gluconate concentrations of 0.01% v/vor below, MRSA reductions were equivalent to that achieved usingilluminated methylene blue alone, indicating that these concentrationsof chlorhexidine were no longer contributing to antimicrobial efficacy.

The data provided in Example II shows that combining low concentrationchlorhexidine gluconate (e.g., above 0.01% v/v) with photo-activatedmethylene blue results in a more powerful short-term antimicrobialeffect of reducing MRSA than using photo-activated methylene blue alone.Several of the concentrations of chlorhexidine used in these studieswere shown to have a measurable anti-microbial effect of reducing MRSAon its own; however it was significantly less than the photodynamicdisinfection method of using the combination of low concentration ofchlorhexidine and methylene blue.

Example III

In vitro experiments were conducted by applying either a control ofpurified water or the following Composition X to planktonic cultures ofS. aureus (Staphylococcus aureus ATCC® 25923™) of approximately 10⁷ to10⁸ CFU/ml. Composition X contained the active ingredients methyleneblue at a concentration of about 0.01% v/v and chlorhexidine digluconateat a concentration of about 0.25% v/v in purified water. Cultures inpurified water or Composition X were left in the dark or irradiatedusing a 670 nm non-thermal laser with a total energy dose of about 20.6Joules/cm² (60 second exposure). After exposure, all samples werediluted and plated on solid media to observe subsequent growth. Thereduction in viability of S. aureus in each experimental condition wascompared to a planktonic S. aureus sample in purified water thatreceived no irradiation (“Control”).

The results showed significant antimicrobial efficacy against S. aureusafter exposure to the irradiated Composition X. The irradiatedComposition X achieved 5.4 log₁₀ reduction in S. aureus viabilitycompared to the Control. Exposure to the non-irradiated Composition Xproduced little reduction in viability of planktonic S. aureus, withabout 0.7 log₁₀ reduction in compared to the Control. This resultindicates that, in the absence of light activation of methylene blue,the antimicrobial efficacy of 0.25% chlorhexidine digluconate after 60second exposure was insignificant. Additionally, the samples in purifiedwater that were irradiated showed no significant reduction in bacterialviability as compared to the Control indicating that the reductioneffect was not due to thermal or light effects from the laser treatmentalone. These results showed that the combination of a photosensitizer(e.g., a phenothiazine such as methylene blue) and chlorhexidinedigluconate had a synergistic effect in providing significantly enhancedantimicrobial efficacy when used for photodynamic disinfection.

Example IV

In vitro experiments were conducted by exposing MRSA(Methicillin-resistant Staphylococcus aureus ATCC® 33592) atapproximately 10⁷ to 10⁸ CFU/ml to a control of purified water or thefollowing compositions. Composition A contained the active ingredientsof methylene blue at a concentration of about 0.01% v/v andchlorhexidine digluconate at a concentration of about 0.25% v/v inpurified water. Composition B contained the active ingredient ofmethylene blue at a concentration of about 0.01% wt in purified water.Composition C contained the active ingredient of chlorhexidinedigluconate at a concentration of about 0.25% v/v in purified water.

MRSA bacterial inocula exposed to methylene blue (Compositions A and B)or purified water were irradiated using a 670 nm non-thermal laser witha total energy dose of 10.3 Joules/cm² (about 30 seconds of exposure).The inocula exposed to chlorhexidine digluconate alone (Composition C)received no irradiation but were left alone for 30 seconds beforeneutralization of the chlorhexidine using Dey-Engley broth. Theneutralizing solution stops the antimicrobial activity of chlorhexidinethus allowing equivalent treatment and/or exposure times to test agentacross all of the experimental samples.

After exposure, all samples were diluted and plated on solid media toobserve subsequent growth. The reduction in viability of MRSA in eachexperimental condition was compared to a planktonic MRSA sample inpurified water that received no irradiation (“Control”).

The results showed that Composition A (methylene blue and chlorhexidinedigluconate) was the most effective treatment for the eradication ofMRSA. Exposure to this composition with irradiation produced a 7.3 log₁₀reduction in MRSA viability (100% eradication) compared to the Control.Irradiation in the presence of Composition B (methylene blue) produced a4.8 log₁₀ reduction in MRSA viability compared to the Control. Exposureto Composition C (chlorhexidine digluconate) produced negligible levelsof eradication with only a 0.4 log₁₀ reduction in MRSA viabilitycompared to the Control. The samples in purified water that wereirradiated showed no significant reduction in bacterial viability ascompared to the Control indicating that the reduction effect was not dueto thermal or light effects from the laser treatment alone. In summary,the antibacterial efficacy of the combined treatment of methylene blueand chlorhexidine digluconate with light irradiation was significantlybetter than that using chlorhexidine digluconate or irradiated methyleneblue alone. This indicates a potentiation effect upon combination ofthese two agents that creates a more powerful antibacterial action thanwould be expected by the simple addition of the reduction effects seenwith each separately.

Example V

In vitro experiments were conducted by exposing MRSA(Methicillin-resistant Staphylococcus aureus ATCC® 33592) atapproximately 10⁷ to 10⁸ CFU/ml to either a control of purified water orthe following compositions. Composition D contained the activeingredient of methylene blue at a concentration of about 0.01% w/v.Composition E contained the active ingredient of chlorhexidinedigluconate at a concentration of about 0.125% v/v in purified water.Composition F contained the active ingredient of chlorhexidinedigluconate at a concentration of about 0.25% v/v in purified water.Composition G contained the active ingredients of methylene blue at aconcentration of about 0.01% w/v and chlorhexidine digluconate at aconcentration of about 0.125% v/v in purified water. Composition Hcontained the active ingredients of methylene blue at a concentration ofabout 0.01% w/v and chlorhexidine digluconate at a concentration ofabout 0.25% v/v in purified water.

All methylene blue containing samples (Compositions D, G and H) wereirradiated using a 670 nm non-thermal laser with a total energy dose of10.3 Joules/cm² (about 30 seconds of exposure). The samples in purifiedwater and chlorhexidine digluconate alone (Compositions E and F)received no irradiation but were left alone for 30 seconds beforeneutralization using Dey-Engley broth. This neutralizing solution stopsthe antimicrobial activity of chlorhexidine, thus allowing equivalenttreatment and/or exposure times to test agent across all of theexperimental samples.

After exposure, all samples were diluted and plated on solid media toobserve subsequent growth. The reduction in viability of MRSA in eachexperimental condition was compared to a planktonic MRSA sample inpurified water that received no irradiation (“Control”)

The results showed that MRSA exposed to Composition D (methylene blue)with irradiation underwent a 4.8 log₁₀ reduction in viability comparedto the Control. Exposure to compositions E and F (chlorhexidinedigluconate) produced no significant reductions in MRSA viabilitycompared to the Control. Exposure to composition G (methylene blue and0.125% chlorhexidine digluconate) with irradiation produced a 3.8 log₁₀reduction in MRSA viability compared to the Control. Exposure tocomposition H (methylene blue and 0.25% chlorhexidine digluconate) withirradiation produced the greatest antibacterial effect against MRSA,with a 7.3 log₁₀ reduction in viability (100% eradication) compared toControl.

Based upon the data described above, the antimicrobial efficacy of thecompositions containing both methylene blue and chlorhexidinedigluconate was significantly better than that achieved with eitheragent alone. Furthermore, the reduction in MRSA viability for thecombined treatment was greater than the combined efficacy of the twoindividual treatments, indicating a potentiation effect. This datasuggests a true potentiation effect, as opposed to simply additiveaction of two different antibacterials, since the concentration ofchlorhexidine digluconate tested alone had no effect on MRSA viabilityafter a 30 second exposure.

As shown in Example I, the native optical absorbance profile ofmethylene blue is not altered in the presence of chlorhexidinedigluconate. Therefore, it is unlikely that the two components complexedor significantly reacted to change the structure of one or the other. Itis more likely that since chlorhexidine is known to act on the outermembrane of Gram-positive organisms, low concentrations that are notbactericidal alone permeabilize the bacterium to photosensitizer. Thiswould allow increased membrane and intracellular aggregation ofmethylene blue molecules. The strong eradication of MRSA achieved bycombining methylene blue with sub-lethal concentrations of chlorhexidinesuggests that this may be a promising formulation for photodynamicdisinfection of MRSA.

Example VI

A study was conducted to determine the efficacy of methylene blue,chlorhexidine digluconate and combinations thereof to eradicate biofilmsof MRSA. Biofilms were grown in flat bottom 96-well culture plates byseeding each well with an inoculum of planktonic MRSA(Methicillin-resistant Staphylococcus aureus ATCC® 33592) atapproximately 10⁸ CFU/ml and allowing growth for 48 hours with shakingat 35° C. to 37° C. After biofilms were established under this protocol,the liquid media was removed from test wells and the wells were rinsedtwice using a phosphate buffered saline solution to remove allplanktonic, non-biofilm associated organisms.

In vitro experiments were conducted by applying 200 μl of each of thefollowing compositions to the biofilms for period of approximately 10seconds. A control of phosphate buffered saline solution. Composition Icontaining the active ingredient of methylene blue at a concentration ofabout 0.01% v/v and chlorhexidine digluconate in a concentration ofabout 0.25% v/v in purified water. Composition J containing the activeingredient of methylene blue at a concentration of about 0.01% v/v inpurified water. Composition K containing the active ingredient ofchlorhexidine digluconate at a concentration of about 0.25% v/v inpurified water. Composition L containing the active ingredient ofchlorhexidine digluconate in a concentration of about 0.50% v/v inpurified water. Composition M containing the active ingredientchlorhexidine digluconate in a concentration of about 0.125% v/v inpurified water. Composition N containing the active ingredient ofmethylene blue at a concentration of about 0.01% v/v and chlorhexidinedigluconate in a concentration of about 0.50% v/v in purified water.Composition O containing the active ingredient of methylene blue at aconcentration of about 0.01% v/v and chlorhexidine digluconate in aconcentration of about 0.125% v/v in purified water.

After 10 seconds, the compositions were all withdrawn from theirrespective the biofilm wells. The biofilm wells treated with methyleneblue (Compositions I, J, N and O) were irradiated with a 670 nmnon-thermal diode laser with a total energy dose of approximately 7Joules/cm² (about 20 seconds of exposure). The biofilm wells exposed tothe purified water composition or one of the chlorhexidine digluconatealone compositions (Compositions K, L and M) were left alone in the darkfor 20 seconds without any irradiation. Immediately after the 20 seconds(with or without irradiation), a neutralizing solution of Dey-Engleybroth was added to all of the biofilm wells in both test and controlconditions. Once all of the biofilms had been exposed to theneutralizing solution, the well plate was transferred to anultrasonicator on high setting for 30 minutes. Followingultrasonication, liquid samples from each well were plated on solidmedia to allow growth of surviving organisms. Plate colony counts weresubsequently performed to determine MRSA eradication compared to thenon-irradiated control of phosphate buffered saline solution(“Control”).

The results showed that exposure to Composition J (methylene blue) withirradiation produced a 2.4 log₁₀ reduction in MRSA viability compared tothe Control. Exposure to composition L (0.50% v/v chlorhexidinedigluconate) produced a 1.3 log₁₀ reduction in MRSA viability comparedto the Control. Exposure to composition K (0.25% v/v chlorhexidinedigluconate) produced a 1.1 log₁₀ reduction in MRSA viability comparedto the Control. Exposure to composition M (0.125% v/v chlorhexidinedigluconate) produced a 0.6 log₁₀ reduction in MRSA viability comparedto the Control. Thus, for the chlorhexidine digluconate onlycompositions (Compositions L, K and M), the data showed a decreasingantimicrobial efficacy against MRSA biofilms as the concentration ofchlorhexidine digluconate decreased from 0.5% v/v to 0.125% v/v.

The results upon exposure to methylene blue and chlorhexidinedigluconate with irradiation were as follows. Composition I (methyleneblue and 0.25% v/v chlorhexidine digluconate) produced a 4.2 log₁₀reduction in MRSA viability compared to the Control. Composition M(methylene blue and 0.50% v/v chlorhexidine digluconate) produced a 4.5log₁₀ reduction in MRSA viability compared to the Control. Composition O(methylene blue and 0.125% v/v chlorhexidine digluconate) produced a 4.3log₁₀ reduction in MRSA viability compared to the Control. These resultsshowed that the combined methylene blue with chlorhexidine digluconatecompositions (Compositions I, N and O) produced superior antimicrobialefficacy compared to compositions containing methylene blue alone orchlorhexidine digluconate alone. Moreover, the reductions in viabilityachieved using the combination compositions were somewhat greater thanthe additive antibacterial effect of the individual components, therebysuggesting a potentiation effect. Furthermore, the antimicrobialefficacy for the combined methylene blue with chlorhexidine digluconatecompositions (Compositions I, N and O) decreased only slightly as thechlorhexidine digluconate concentration decreased from 0.5% v/v to0.125% v/v.

Example VII

This study was designed to assess the antibacterial efficacy ofphotodynamic disinfection, using various photosensitizer compositions,on human full thickness skin cultures colonized on the epithelialsurface with high levels of MRSA.

Stock vials of MRSA (Methicillin-resistant Staphylococcus aureus ATCC®33592) were kept frozen at −80° C. before use. Upon thawing, cultureswere plated on tryptic soy agar (Hardy Diagnostics located in SantaMaria, Calif.) and grown at 37° C. until colonies were visible. Thesewere sub-cultured to ensure growth phase and used to create inocula of˜10⁹ CFU/ml for colonization of skin surfaces.

The human skin culture model used for this study was the EpiDerm FT™Full Thickness Skin Model (MatTek™ Corporation located in Ashland,Mass.). This product consists of human-derived epidermal keratinocytesand human-derived dermal fibroblasts cultured at an air/media interfaceto form a stratified (epidermis and dermis), intact model of fullthickness epithelialized human skin. These structures have been shown toexhibit differentiation markers, lipid profiles, and basement membranestructure characteristic of the in vivo situation and have been usedextensively to study the effects of agents/treatments on human skin.Skin samples were received in cell culture inserts in 6-well plates fromthe manufacturer, and placed in culture at 37° C. (5% CO₂) for 24 hoursafter shipping to allow equilibration before use. A small volume (25 μl)of MRSA inocula, prepared as described above, was pipetted onto to theapical surface of the culture sample, taking care not to overflow to thesides of the insert, and incubated overnight at 37° C. (5% CO₂). Sterilecotton-tipped swabs were used to sample the inoculated tissue surfacesevery 24 hours for 5 days post-inoculation in order to confirm stablecolonization. Data showed that inoculating the epithelial surface ofhuman skin cultures with 10⁹ CFU/ml of MRSA resulted in a stablecolonization of ˜10⁷ CFU/ml (recoverable organisms after swab sampling)over a 5 day period.

Experiments were conducted by applying a control of purified water orone of the following compositions to the epithelial surface of MRSAcolonized skin structures. Each of the samples of skin structuresreceived a 50 μl aliquot of one of the following compositions: (i) acontrol of purified water (“Control”); (ii) Composition P contained theactive ingredients of methylene blue at a concentration of about 0.01%v/v and chlorhexidine digluconate at a concentration of about 0.25% v/vin purified water; (iii) Composition Q contained the active ingredientof methylene blue at a concentration of about 0.01% wt in purifiedwater; and (iv) Composition R contained the active ingredient ofchlorhexidine digluconate at a concentration of about 0.25% v/v inpurified water.

After application of either purified water, Composition P, Composition Qor Composition R, skin structures were placed directly under a 670 nmfiber-optically coupled laser system, which was terminated at anSMA-type connector and suspended using a laboratory stand/clamp. Thetissue of each sample was placed at a distance of 7 cm from theterminating end of the fiber-optic source in order to produce a powerdensity at the tissue surface equivalent to that of the surface of theMRSAid™ light diffuser tip manufactured by Ondine Biopharma Corp.located at Vancouver, B.C., Canada (˜400 mW/cm²). The samples wereirradiated by a 670 nm non-thermal diode laser using this power densityfor about 120 seconds (total energy dose=about 48 Joules/cm²). Thismethod of irradiation was necessary since the MRSAid™ light diffuser tipitself could not be placed into the cell culture insert due toincompatibilities in size and shape.

After the 120 seconds, the samples received another 50 μl application oftheir respective composition (i.e., purified water, Composition P,Composition Q, or Composition R) and another round of irradiation for120 seconds (total energy dose=about 48 Joules/cm²) using the sameprocess as described above. Thus, the samples received a total energydose of 96 Joules/cm² from the two rounds of irradiation.

Immediately after the second round of irradiation, excess compositionwas removed from the treated surface of the samples. Sterilecotton-tipped swabs were used to sample the tissue surface, and thesewere neutralized to inhibit the action of chlorhexidine digluconateusing a 0.45% v/v saline solution containing 3% tween-80 and 0.75%lecithin. Preliminary experiments confirmed that this solutioneffectively neutralized any chlorhexidine digluconate present on theswab before plating for viability assessment. Swabs were placed inliquid recovery media and samples were plated on solid media to observesubsequent growth. The reduction in viability of MRSA in eachexperimental condition was compared to a planktonic MRSA sample inpurified water that received no irradiation (“Control”)

Half of the treated skin structures were sampled immediately aftertreatment and the other half were incubated for 24 hours beforesampling. Data from samples taken immediately post-treatment showed: (i)Exposure to composition Q (methylene blue) with irradiation resulted ina 0.2 log₁₀ reduction in MRSA viability compared to the Control; (ii)Exposure to composition C (chlorhexidine digluconate) produced a 1.1log₁₀ reduction in MRSA viability compared to the Control; and (iii)Exposure to composition P (methylene blue and chlorhexidine digluconate)resulted in a 5.1 log₁₀) reduction in MRSA viability compared to theControl. This immediate sampling data showed that the combination ofmethylene blue and chlorhexidine digluconate (Composition P) resulted ina significant and rapid reduction in MRSA viability when irradiated. Incontrast, application of methylene blue alone (Composition Q) or lightalone (irradiated control with purified water) did not result insignificant reductions in viability compared to the Control immediatelypost-treatment.

Data from samples taken 24 hours post-treatment showed: (i) Exposure tocomposition Q (methylene blue) was equivalent to that of the Controlimmediately post-treatment; (ii) Exposure to composition R(chlorhexidine digluconate) produced a 4.3 log₁₀ reduction in MRSAviability compared to the Control, which was significantly greater thanthat observed immediately post-treatment; and (iii) Exposure tocomposition P (methylene blue and chlorhexidine digluconate) resulted ina 5.9 log₁₀ reduction in MRSA viability compared to the Control, whichrepresented near total eradication of all colonized MRSA on that tissuesurface.

These results indicate that Composition P, containing both methyleneblue and chlorhexidine digluconate, was more effective at reducingcolonization of MRSA on skin surfaces than the single ingredientcompositions both immediately after treatment and at 24 hourspost-treatment. The combination of a photosensitizer (e.g., aphenothiazine such as methylene blue) and chlorhexidine digluconatepotentiated the reduction effect in comparison to the sum of thatachieved with either agent alone.

Example VIII

A second study was conducted using the skin culture model described inExample VII to determine long term suppression of MRSA growth. Either acontrol of purified water or Composition S containing the activeingredients of methylene blue at a concentration of about 0.01% v/v andchlorhexidine digluconate at a concentration of about 0.25% v/v inpurified water were applied to MRSA colonized skin samples and treatedusing the same application, irradiation, and sampling protocols andprocedures described in EXAMPLE VII. Surface swab samples were taken at24 hours, 48 hours and 120 hours post-treatment. Due to constraints onthe number of samples available for testing, swab samples were not takenimmediately post-treatment for this study.

At 24 hours post-treatment, exposure to composition S with irradiationresulted in a 3.6 log₁₀ reduction in MRSA viability compared to thenon-irradiated control (purified water). At 48 hours post-treatment andat 120 hours post-treatment, exposure to composition S resulted in totaleradication of MRSA. In corresponding non-treated controls at those timepoints, bacterial viability remained in the 10⁷-10⁸ CFU/ml range. Thisshowed that the loss of bacterial viability in the treatment conditionwas not due to natural loss of colonization or decrease in tissueviability in the cultures.

Example IX

A third study was conducted using the skin culture model described inExample VII to determine long term suppression of MRSA growth. Either acontrol of purified water or one of the following compositions wereapplied to skin samples and treated using the same application,irradiation, and sampling protocols and procedures described in EXAMPLEVII. Composition T contained the active ingredient of methylene blue ata concentration of about 0.01% v/v and chlorhexidine digluconate in aconcentration of about 0.25% v/v in purified water. Composition Ucontained the active ingredient of methylene blue at a concentration ofabout 0.01% v/v in purified water. Swab samples were taken (i)immediately after photodynamic disinfection treatment as described inEXAMPLE VII; (ii) at 24 hours post-treatment; and (iii) at 48 hourspost-treatment.

The results showed that for samples taken immediately post-treatment,exposure to composition T (methylene blue and chlorhexidine digluconate)with irradiation produced a 1.1 log₁₀ reduction in MRSA viabilitycompared to its comparable non-irradiated control. For the 24 hourspost-treatment samples, exposure to composition T with irradiationproduced a 3.1 log₁₀ reduction in MRSA viability compared to itscomparable non-irradiated control. For the 48 hours post-treatmentsamples, exposure to composition T with irradiation produced a 3.5 log₁₀reduction in MRSA viability compared to its comparable non-irradiatedcontrol.

Furthermore, in the non-irradiated controls, the colonized MRSA countsincreased over time with log₁₀ counts of (i) 6.8 for the immediatepost-treatment samples; (ii) 7.8 for the 24 hours post-treatmentsamples; and (iii) 8.4 for the 48 hours post-treatment samples.

Finally, the results showed that exposure to Composition U (methyleneblue) with irradiation resulted in no significant reduction of MRSAviability compared to non-irradiated controls at any of the time pointstested. These results showed that the combination of a methylene blueand chlorhexidine digluconate had a potentiation effect that providedsignificantly enhanced antimicrobial efficacy; suppressing MRSA growthover a 48 hour period.

What is claimed is:
 1. A method of treating Methicillin-resistantStaphylococcus aureus (“MRSA”) comprising: (a) applying aphotosensitizing composition to treatment site comprising: aphenothiazine at a concentration from about 0.001% wt to about 1% wt;chlorhexidine at a concentration from about 0.125% v/v to about 0.5%v/v; and a pharmaceutically acceptable carrier; and (b) applying lightto the treatment site at a wavelength absorbed by the photosensitizingcomposition so as to reduce the MRSA located at the treatment site. 2.The method of claim 1 wherein the phenothiazine is methylene blue. 3.The method of claim 1 wherein the phenothiazine is toluidine blue. 4.The method of claim 1 wherein chlorhexidine is chlorhexidinedigluconate.
 5. The method of claim 1 wherein concentration of thechlorhexidine is between about 0.25% v/v and about 0.5% v/v.
 6. Thecomposition of claim 1 wherein concentration of the phenothiazine isbetween about 0.01% wt to about 1% wt.
 7. The method of claim 1 whereinthe light applied to the treatment site in step (b) includes multiplewavelengths that are absorbed by the photosensitizing composition. 8.The method of claim 1 wherein the treatment site is the nasal cavity. 9.The method of claim 1 wherein the treatment site is anterior nasalnares.
 10. A method for photodynamic disinfection of microbescomprising: (a) applying a photosensitizing composition to treatmentsite comprising: a phenothiazine at a concentration from about 0.001% wtto about 1% wt; chlorhexidine at a concentration from about 0.125% v/vto about 0.5% v/v; and a pharmaceutically acceptable carrier; and (b)applying light to the treatment site at a wavelength absorbed by thephotosensitizing composition so as to reduce the microbes located at thetreatment site.
 11. The method of claim 10 wherein the phenothiazine ismethylene blue.
 12. The method of claim 10 wherein the phenothiazine istoluidine blue.
 13. The method of claim 10 wherein chlorhexidine ischlorhexidine digluconate.
 14. The method of claim 10 whereinconcentration of the chlorhexidine is between about 0.25% v/v and about0.5% v/v.
 15. The method of claim 10 wherein concentration of thephenothiazine is between about 0.01% wt to about 0.1% wt.
 16. The methodof claim 10 wherein the light applied to the treatment site in step (b)includes multiple wavelengths that are absorbed by the photosensitizingcomposition.
 17. The method of claim 10 wherein the treatment site isthe nasal cavity.
 18. The method of claim 10 wherein the microbes areselected from the group consisting of Methicillin-resistantStaphylococcus aureus, Staphylococcus aureus, Escherichia coli,Enterococcus faecalis, Pseudomonas aeruginosa, Aspergillus, Candida,Clostridium difficile, Staphylococcus epidermidis, Acinetobacter sp.,Porphyromonas, Prevotella, Fusobacterium, Tannerella, Actinobacillus,Selenomonas, Eikenella, Campylobacter, Wolinella and a combinationthereof.
 19. A method for photodynamic disinfection of microbescomprising: (a) applying a photosensitizing composition to treatmentsite comprising: an effective amount of phenothiazine; an effectiveamount of chlorhexidine; and a pharmaceutically acceptable carrier; and(b) applying light to the treatment site at a wavelength absorbed by thephotosensitizing composition so as to reduce the microbes located at thetreatment; wherein the effective amount of phenothiazine and theeffective amount of chlorhexidine produces a potentiation effect ofantimicrobial efficacy during the photodynamic disinfection of microbes.20. The method of claim 19 wherein the phenothiazine is methylene blue.21. The method of claim 19 wherein the phenothiazine is toluidine blue.22. The method of claim 19 wherein chlorhexidine is chlorhexidinedigluconate.
 23. The method of claim 19 wherein concentration of thechlorhexidine is between about 0.25% v/v and about 0.5% v/v.
 24. Thecomposition of claim 19 wherein concentration of the phenothiazine isbetween about 0.01% wt to about 1% wt.
 25. The method of claim 19wherein the light applied to the treatment site in step (b) includesmultiple wavelengths that are absorbed by the photosensitizingcomposition.
 26. The method of claim 19 wherein the microbes includesMethicillin resistant Staphylococcus aureus.
 27. The method of claim 19wherein the treatment site is the nasal cavity.
 28. The method of claim19 wherein the microbes are selected from the group consisting ofMethicillin-resistant Staphylococcus aureus, Staphylococcus aureus,Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa,Aspergillus, Candida, Clostridium difficile, Staphylococcus epidermidis,Acinetobacter sp., Porphyromonas, Prevotella, Fusobacterium, Tannerella,Actinobacillus, Selenomonas, Eikenella, Campylobacter, Wolinella and acombination thereof.